PROJECT SUMMARY Dengue virus (DENV) is the most important mosquito-borne viral disease affecting humans today, with an estimated 2.5 billion living in areas at risk for epidemic transmission. Despite this, there are presently no antiviral drugs effective against dengue virus nor is there a protective vaccine. Viral infection can be regarded as a coordinated series of exogenous biochemical reactions that integrate with the biochemical equilibria of the host cell. The specific cellular factors that support dengue virus infection have, for the most part, not been identified. Likewise, the proximal and distal biochemical pathways that impinge on the dengue virus replication cycle are unknown. Small molecules that interact with specific cellular targets have become a powerful way to probe cellular pathways and may provide an important means by which to characterize the biochemical pathways and potential pharmacological intervention points associated with dengue virus infection of the host cell. This proposal aims to develop methods for identifying small molecules that can be used to probe interactions between DENV and its host cell in a kinetically reversible manner. We have designed a system in which fluorescence resonance energy transfer (FRET) is used to monitor activity of a dengue viral enzyme as a marker for viral infection, gene expression, replication, and spread in host cells. The goal of this proposal is to develop this system into a high-throughput cellular assay that enables us to identify compounds that interfere with the dengue virus-host cell infection cycle. The optimized assay will be used to screen a library of known bioactives. We will then use RNA interference to confirm that the anti-dengue effects of screening "hits" are indeed mediated by the cellular factors known to be targeted by members of the compound collection. In addition, we will use a panel of secondary and tertiary assays to validate "hits" from the screen. This will include (1) measurement of compound cytotoxicity in the presence and absence of dengue virus infection; (2) measurement of effects on infectious dengue viral titers; (3) measurement of effects on viral protein translation and processing; (4) measurement of effects on viral genome replication; (5) ultrastructural characterization of compound effects on dengue-infected and mock- infected cells by electron microscopy; and (6) characterization of the viral specificity of anti-dengue compounds by measuring their effects against a panel of viruses that include isolates representative of all four DENV serotypes, as well as additional flaviviruses and viruses beyond the Flaviviridae family. Collectively, these experiments will provide tools that enable the reliable detection of small molecules that can be used to probe the interactions between dengue virus and its host cell at the molecular level. The general strategy may also be extended to examine interactions between other viruses and their hosts. Project Narrative Dengue virus infects 100 million people and causes up to half a million cases of potentially fatal dengue hemorrhagic fever annually; yet there are currently no antiviral drugs effective against dengue virus, nor is there a protective vaccine. All viruses replicate inside a host cell and steal resources in order to survive, but we know relatively little about how dengue interacts with its host-what it needs or how it causes disease. We are developing tools that enable us to identify small, drug-like molecules that can inhibit the virus by interacting with targets in the host cell. These inhibitors can be used to study how dengue virus interacts with its host, and they may also be useful in developing therapies for the treatment of dengue virus infections. [unreadable] [unreadable] [unreadable]